Mucin-type glycosylation is one of the most prevalent posttranslational modifications of proteins and plays an important role in many biological processes. Mucin and mucin-like glycoproteins are potential cancer markers. Glycoproteins are usually isolated as heterogeneous glycoforms from natural sources, and this complicates studies of their structure and function and hinders the development of their affinity capturing reagents. Current methods for synthesis of glycoproteins are time consuming and technically demanding. The goal of this project is to establish a cell-free translation system that can simply and economically produce glycoproteins with defined O-glycan structures. O-Glycans will be incorporated into proteins through translational suppression at desired site(s). The main objective of the project is to develop amino acyl-tRNA synthetases (aaRS) that are able to activate glycoamino acids carrying structurally defined O-glycans. Phase I will focus on construction mutant aaRS libraries and establish methods for selecting synthetases for a diglycoamino acid. Phase II will continue the effort and refine the screen process for glycoamino acids with various glycan structures. The screen will also extend to tri- or tetra-glycoamino acid in Phase II. These selected synthetases will be used in high yield cell-free trans lation system to produce desired 0- glycoprotein. In phase I, the objective is to develop an E. coli cell-free expression system that is able to produce protein containing T -antigens in a yield up to 0.1 mg per 1 ml of translation reaction.